EP4041802A1 - Composition polymère - Google Patents

Composition polymère

Info

Publication number
EP4041802A1
EP4041802A1 EP20793432.4A EP20793432A EP4041802A1 EP 4041802 A1 EP4041802 A1 EP 4041802A1 EP 20793432 A EP20793432 A EP 20793432A EP 4041802 A1 EP4041802 A1 EP 4041802A1
Authority
EP
European Patent Office
Prior art keywords
optionally substituted
mol
composition
rna
alkenyl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20793432.4A
Other languages
German (de)
English (en)
Inventor
Robin Shattock
Molly Stevens
Yunqing ZHU
Anna Blakney
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ip2ipo Innovations Ltd
Original Assignee
Imperial College Innovations Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Imperial College Innovations Ltd filed Critical Imperial College Innovations Ltd
Publication of EP4041802A1 publication Critical patent/EP4041802A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L79/00—Compositions of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing nitrogen with or without oxygen or carbon only, not provided for in groups C08L61/00 - C08L77/00
    • C08L79/02—Polyamines
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/02—Polyamines
    • C08G73/0246—Polyamines containing other atoms than carbon, hydrogen, nitrogen or oxygen in the main chain
    • C08G73/0253—Polyamines containing sulfur in the main chain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/39—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton at least one of the nitrogen atoms being part of any of the groups, X being a hetero atom, Y being any atom
    • C07C323/40—Y being a hydrogen or a carbon atom
    • C07C323/41—Y being a hydrogen or an acyclic carbon atom
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/02—Polyamines
    • C08G73/028—Polyamidoamines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration

Definitions

  • the present invention relates to polymeric compositions, nanoparticles and vaccines comprising polymeric compositions.
  • the invention extends to medical uses of the polymeric compositions, nanoparticles and vaccines.
  • the invention further extends to methods of producing the polymeric compositions and nanoparticles.
  • mRNA messenger RNA
  • saRNA Self-amplifying mRNA (saRNA), derived from the alphavirus genome, 8 is particularly advantageous as a vaccine platform, as it self-replicates upon delivery into the cytoplasm which results in augmented protein expression and a minimum required dose of RNA.
  • saRNA is a relatively large (-9,500 nt), negatively charged molecule, it requires a delivery vehicle for efficient cellular uptake.
  • saRNA has previously been delivered using cationic emulsions, 12 lipid nanoparticles, 10 and polymers. 9 ’ 13
  • these delivery platforms were initially developed and optimized for shorter nucleic acids, such as siRNA (-20 nt) and mRNA (-2,000-5,000 nt) and thus may not be the optimal formulation for saRNA.
  • siRNA 20 nt
  • mRNA -2,000-5,000 nt
  • a polymeric composition comprising a plurality of polymers of formula (I): , wherein L 1 to ⁇ J> are each independently an optionally substituted C - 2 alkylene, an optionally substituted C 2-12 alkenylene, an optionally substituted C 2-i2 alkynylene, an optionally substituted C 3-6 cycloalkylene, an optionally substituted 3 to 8 membered heterocyclylene, an optionally substituted Cc,- 12 arylene, an optionally substituted 5 to 10 membered heteroarylene or L 6 L 7 , wherein adjacent carbon atoms in the alkylene, alkenylene or alkynylene are optionally interrupted by one or more heteroatoms;
  • L 6 and L 7 are independently an optionally substituted CV 12 alkylene, an optionally substituted C 2-i2 alkenylene, an optionally substituted C 2-i2 alkylnyene, an optionally substituted C 3-6 cycloalkylene, an optionally substituted 3 to 8 membered heterocyclylene, an optionally substituted Cc,- 12 arylene or an optionally substituted 5 to 10 membered heteroarylene, wherein adjacent carbon atoms in the alkylene, alkenylene or alkynylene are optionally interrupted by one or more heteroatoms;
  • R 1 and R 2 are each independently H, an optionally substituted CV 12 alkyl, an optionally substituted C 2-i2 alkenyl or an optionally substituted C 2-i2 alkynyl;
  • R3 is -OR 4 , -COOR 4 , -S0 2 0R 4 , (0CH 2 CH 2 ) m 0H, or NR 4 Rs,
  • R 4 and R 5 are each independently H, an optionally substituted C - i2 alkyl, an optionally substituted C 2-i2 alkenyl, an optionally substituted C 2-i2 alkynyl, an optionally substituted C 3-6 cycloalkyl, an optionally substituted 3 to 8 membered heterocyclyl, an optionally substituted C - 12 aryl or an optionally substituted 5 to 10 membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or alkenyl are optionally interrupted by one or more heteroatoms; and m is an integer between 1 and 10; or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof; characterised in that the average molecular mass of the plurality of polymers of formula (I) is greater than 5 kg mol ⁇ 1 .
  • the inventors have been able to synthesise pABOLs with higher molar masses than was possible using prior art methods.
  • alkyl refers to a saturated straight or branched hydrocarbon.
  • the alkyl group is a primary, secondary, or tertiary hydrocarbon.
  • the alkyl group includes one to six carbon atoms, i.e. C 1 -C 6 alkyl.
  • C 1 -C 6 alkyl includes for example methyl, ethyl, n-propyl (l-propyl) and isopropyl (2-propyl, l-methylethyl), butyl, pentyl, hexyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, and isohexyl.
  • Alkenyl refers to olefmically unsaturated hydrocarbon groups which can be unbranched or branched.
  • the alkenyl group has 2 to 6 carbons, i.e. it is a C 2 -C 6 alkenyl.
  • C 2 -C 6 alkenyl includes for example vinyl, allyl, propenyl, butenyl, pentenyl and hexenyl.
  • Alkynyl refers to acetylenically unsaturated hydrocarbon groups which can be unbranched or branched.
  • the alkynyl group has 2 to 6 carbons, i.e. it is a C 2 -C 6 alkynyl.
  • C 2 -C 6 alkynyl includes for example propargyl, propynyl, butynyl, pentynyl and hexynyl.
  • alkylene refers to a bivalent saturated straight or branched hydrocarbon.
  • the alkylene group is a primary, secondary, or tertiary hydrocarbon.
  • the alkylene group includes one to six carbon atoms, i.e. C 1 -C 6 alkylene.
  • C 1 -C 6 alkylene includes for example methylene, ethylene, n-propylene and isopropylene, butylene, pentylene, hexylene, isobutylene, sec-butylene, tert-butylene, isopentylene, neopentylene, and isohexylene.
  • alkenylene refers to a bivalent olefmically unsaturated straight or branched hydrocarbon.
  • the alkenylene group is a primary, secondary, or tertiary hydrocarbon.
  • the alkenylene group includes one to six carbon atoms, i.e. C 2 -C 6 alkenylene.
  • C 2 -C 6 alkenylene includes for example ethenylene, propenylene, butenylene, pentenylene or hexenylene.
  • alkynylene refers to a bivalent acetylenically unsaturated straight or branched hydrocarbon.
  • the alkynylene group is a primary, secondary, or tertiary hydrocarbon.
  • the alkynylene group includes one to six carbon atoms, i.e. C 2 -C 6 alkynylyne.
  • C 2 -C 6 alkynylene includes for example ethynylene, propynylene, butynylene, pentynylene or hexynylene.
  • Cycloalkyl refers to a non-aromatic hydrocarbon 3 to 6 membered ring system.
  • the cycloalkyl may be saturated or partially saturated and may be monocyclic, bicyclic or polycyclic.
  • the cycloalkylene may be saturated or partially saturated and may be monocyclic, bicyclic or polycyclic.
  • Representative examples of a C 3 -C 6 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.
  • Cycloalkylene refers to a bivalent, non-aromatic, hydrocarbon 3 to 6 membered ring system.
  • the cycloalkylene may be saturated or partially saturated and may be monocyclic, bicyclic or polycyclic.
  • Heterocycle or “heterocyclyl” refers to a 3 to 8 membered monocyclic, bicyclic or bridged molecules in which at least one ring atom is a heteroatom.
  • the or each heteroatom may be independently selected from the group consisting of oxygen, sulfur and nitrogen.
  • a heterocycle may be saturated or partially saturated.
  • Exemplary 3 to 8 membered heterocyclyl groups include but are not limited to aziridine, oxirane, oxirene, thiirane, pyrroline, pyrrolidine, dihydrofuran, tetrahydrofuran, dihydrothiophene, tetrahydrothiophene, dithiolane, piperidine, 1, 2,3,6- tetrahydropyridine-i-yl, tetrahydropyran, pyran, morpholine, piperazine, thiane, thiine, piperazine, azepane, diazepane, oxazine.
  • Heterocyclylene refers to a 3 to 8 membered bivalent monocyclic, bicyclic or bridged molecules in which at least one ring atom is a heteroatom. The or each heteroatom may be independently selected from the group consisting of oxygen, sulfur and nitrogen.
  • Aryl refers to an aromatic 6 to 12 membered hydrocarbon group. Examples of a C 6 -C 12 aryl group include, but are not limited to, phenyl, a-naphthyl, b-naphthyl, biphenyl, tetrahydronaphthyl and indanyl.
  • Allene refers to a bivalent aromatic 6 to 12 membered hydrocarbon group.
  • Heteroaryl refers to a monocyclic or bicyclic aromatic 5 to 10 membered ring system in which at least one ring atom is a heteroatom.
  • the or each heteroatom may be independently selected from the group consisting of oxygen, sulfur and nitrogen.
  • Examples of 5 to 10 membered heteroaryl groups include furan, thiophene, indole, azaindole, oxazole, thiazole, isoxazole, isothiazole, imidazole, N-methylimidazole, pyridine, pyrimidine, pyrazine, pyrrole, N-methylpyrrole, pyrazole, N-methylpyrazole, 1,3,4-oxadiazole, 1,2,4-triazole, 1- methyl-i, 2, 4-triazole, lH-tetrazole, l-methyltetrazole, benzoxazole, benzothiazole, benzofuran, benzisoxazole, benzimidazole, N- methylbenzimidazole, azabenzimidazole, indazole, quinazoline, quinoline, and isoquinoline.
  • Bicyclic 5 to 10 membered heteroaryl groups include those where a phenyl, pyridine, pyrimidine, pyrazine or pyridazine ring is fused to a 5 or 6-membered monocyclic heteroaryl ring.
  • Heteroarylene refers to a bivalent monocyclic or bicyclic aromatic 5 to 10 membered ring system in which at least one ring atom is a heteroatom.
  • the or each heteroatom may be independently selected from the group consisting of oxygen, sulfur and nitrogen.
  • alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, cycloalkyl, cycloalkylene, heterocyclyl, heterocyclylene, aryl, arylene, hereoaryl and heteroarylene groups can be unsubstituted or substituted with one or more of halogen, OR 14 , NR ⁇ R ⁇ , C - 6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3 to 8 membered heterocyclyl, C ⁇ ,- 2 aryl or 5 to 10 membered heteroaryl, where R 14 and R 15 are independently C - 6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3 to 8 membered heterocyclyl, C ⁇ ,- 2 aryl or 5 to 10 membered heteroaryl.
  • a halogen may be fluorine, chlorine, bromine or iodine.
  • L 1 to L 4 are each independently an optionally substituted C - 6 alkylene, an optionally substituted C 2-6 alkenylene or an optionally substituted C 2-6 alkynylene, and more preferably L 1 to L 4 are each independently an optionally substituted C - 3 alkylene, an optionally substituted C 2-3 alkenylene or an optionally substituted C 2-3 alkynylene.
  • the alkylene, alkenylene or alkynylene may be substituted with a halogen.
  • the alkylene, alkenylene or alkynylene is unsubstituted.
  • L 1 to L 4 are each -CH 2 CH 2 -.
  • L 5 is an optionally substituted C 2- s alkylene, an optionally substituted C 2- s alkenylene or an optionally substituted C 2- s alkynylene, and more preferably L 5 is an optionally substituted C 3-5 alkylene, an optionally substituted C 3-5 alkenylene or an optionally substituted C 3-5 alkynylene.
  • the alkylene, alkenylene or alkynylene may be substituted with a halogen.
  • the alkylene, alkenylene or alkynylene is unsubstituted.
  • L 5 is -CH 2 CH 2 CH 2 CH 2 -.
  • R 1 and R 2 are independently H, an optionally substituted C -6 alkyl, an optionally substituted C 2 -6 alkenyl or an optionally substituted C 2 -6 alkynyl, and more preferably are independently H, an optionally substituted C - 3 alkyl, an optionally substituted C 2-3 alkenyl or an optionally substituted C 2-3 alkynyl.
  • the alkyl, alkenyl or alkynyl may be substituted with a halogen.
  • the alkyl, alkenyl or alkynyl is unsubstituted.
  • R 1 and R 2 are H.
  • R3 is preferably NR4R5.
  • R 4 and Rs are independently H, an optionally substituted C -6 alkyl, an optionally substituted C 2 -6 alkenyl or an optionally substituted C 2 -6 alkynyl, and more preferably are independently H, an optionally substituted C - 3 alkyl, an optionally substituted C 2-3 alkenyl or an optionally substituted C 2-3 alkynyl.
  • the alkyl, alkenyl or alkynyl may be substituted with a halogen.
  • the alkyl, alkenyl or alkynyl is unsubstituted.
  • R 4 and R 5 are H.
  • the plurality of polymers of formula (I) maybe a plurality of polymers of formula (la): or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof.
  • the plurality of polymers of formula (I) or (la) may be a pharmaceutically acceptable salt.
  • salt may be understood to refer to any salt of a compound provided herein which retains its biological properties and which is not toxic or otherwise undesirable for pharmaceutical use. Such salts may be derived from a variety of organic and inorganic counter-ions well known in the art.
  • Such salts include, but are not limited to: (1) acid addition salts formed with organic or inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic, acetic, adepic, aspartic, trifluoroacetic, trichloroacetic, propionic, hexanoic, cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic, succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric, benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic, phthalic, lauric, methanesulfonic, ethanesulfonic, 1,2-ethane-disulfonic, 2- hydroxyethanesulfonic, benzenesulfonic, 4-chlorobenzenesulfonic, 2- n
  • Pharmaceutically acceptable salts may include, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium and the like, and when the compound contains a basic functionality, salts of non-toxic organic or inorganic acids, such as hydrohalides, e.g.
  • Hemisalts of acids and bases may also be formed, for example, hemisulphate salts.
  • the plurality of polymers of formula (I) maybe hydrochloric salts. Accordingly, the plurality of polymers of formula (la) may be a plurality of polymers of formula (Iai):
  • solvate maybe understood to refer to a compound provided herein or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. Where the solvent is water, the solvate is a hydrate.
  • the molecular mass maybe characterized using an Agilent PL GPC-50 instrument, equipped with a refractive index (RI) detector, with HPLC grade DMF (containing 0.075 wt% LiBr) as the eluent at a flow rate of 1.0 mL min 1 at 40 °C.
  • RI refractive index
  • Two GRAM Linear columns maybe used in series.
  • Near monodispersed poly( methyl methacrylate) standards may be used to calibrate the instrument.
  • the composition comprising the plurality of polymers of formula (I) may be dissolved in HPLC grade DMF, containing 0.075 wt% LiBr, and filtered through 0.2 pm syringe filters prior to analysis.
  • the average molecular mass of the plurality of polymers of formula (I) may be at least 5.5 kg mol ⁇ 1 , at least 6 kg mol ⁇ 1 , at least 7 kg mol ⁇ 1 , at least 8 kg mol ⁇ 1 or at least 9 kg mol ⁇ 1 .
  • the average molecular mass of the plurality of polymers of formula (I) maybe at least to kg mol ⁇ 1 , at least 20 kg mol ⁇ 1 , at least 30 kg mol ⁇ 1 , at least 40 kg mol ⁇ 1 or at least 50 kg mol ⁇ 1 .
  • the average molecular mass of the plurality of polymers of formula (I) maybe at least 75 kg mol ⁇ 1 , at least too kg mol ⁇ 1 , at least 125 kg mol ⁇ 1 , at least 150 kg mol ⁇ 1 or at least 160 kg mol ⁇ 1 .
  • the average molecular mass of the plurality of polymers of formula (I) may be between 5.5 and 1,000 kg mol ⁇ 1 , between 5.5 and 900 kg mol ⁇ 1 , between 6 and 800 kg mol ⁇ 1 , between 7 and 700 kg mol ⁇ 1 , between 8 and 600 kg mol ⁇ 1 or between 9 and 500 kg mol ⁇ 1 .
  • the average molecular mass of the plurality of polymers of formula (I) may be between 10 and 450 kg mol ⁇ 1 , between 20 and 400 kg mol ⁇ 1 , between 30 and 350 kg mol ⁇ 1 , between 40 and 300 kg mol ⁇ 1 or between 50 and 250 kg mol ⁇ 1 .
  • the average molecular mass of the plurality of polymers of formula (I) maybe between 75 and 225 kg mol ⁇ 1 , between 100 and 200 kg mol ⁇ 1 , between 125 and 190 kg mol ⁇ 1 , between 150 and 180 kg mol ⁇ 1 or between 160 and 170 kg mol ⁇ 1 .
  • the average molecular mass of the plurality of polymers of formula (I) between 5.5 and 100 kg mol ⁇ 1 , between 6 and 50 kg mol ⁇ 1 , between 6.5 and 25 kg mol ⁇ 1 , between 7 and 20 kg mol ⁇ 1 , between 7.5 and 15 kg mol ⁇ 1 , between 7.7 and 10 kg mol ⁇ 1 , between 7.8 and 9 kg mol ⁇ 1 , or between 7.9 and 8.5 kg mol ⁇ 1 .
  • pABOL with an average molecular mass of about 8 kg mol ⁇ 1 is surprisingly effective at delivering self-amplifying RNA (saRNA).
  • n may be an integer. However, n may vary within the plurality of polymers of formula (I). Accordingly, an average n value may be calculated for the plurality of polymers. It may be appreciated that the average n value may be calculated based upon the values of a, b, c, d and e and the average molecular mass. For instance, if the plurality of polymers are polymers of formula (Iai) and the average molecular mass is 8 then the average n value would be about 21.
  • the average n value maybe at least 13, at least 14, at least 16, at least 18, at least 20 or at least 22.
  • the average n value maybe at least 25, at least 50, at least 75, at least too or at least 125.
  • the average n value maybe at least 200, at least 250, at least 300, at least 350 or at least 400.
  • the average n value maybe between 13 and 2500, between 14 and 2400, between 16 and 2200, between 18 and 2000, between 20 and 1750 or between 22 and 1500.
  • the average n value maybe between 25 and 1250, between 50 and 1100, between 75 and 1000, between 100 and 850 or between 125 and 700.
  • the average n value maybe between 200 and 600, between 250 and 550, between 300 and 500, between 350 and 475 or between
  • the average n value maybe between 13 and 450, between 14 and 250, between 15 and 125, between 16 and 75, between 17 and 50, between 18 and 40, between 19 and 30, between 20 and 25 or between 20.3 and 22.5.
  • a composition of matter comprising the polymeric composition according to the first aspect and a nucleic acid.
  • the weight ratio of polymeric composition and the nucleic acid may be between 1: 1 and 200:1, more preferably between 5:1 and 150:1 or between 10:1 and 100:1, and most preferably between 20:1 and 90:1, between 30:1 and 80:1, between 40:1 and 70:1 or between 45:1 and 60:1.
  • the nucleic acid may be DNA, RNA or a DNA/RNA hybrid sequence.
  • the nucleic acid is DNA or RNA.
  • the nucleic acid is RNA.
  • the RNA maybe single stranded or double stranded.
  • the RNA maybe selected from the group consisting of: messenger RNA (mRNA), micro RNA (miRNA); short interfering RNA (siRNA); short hairpin RNA
  • RNA self-amplifying RNA
  • interference RNA interference RNA
  • small RNA small RNA.
  • the RNA is self-amplifying RNA (saRNA).
  • RNAs may contain the basic elements of mRNA (a cap, 5’ UTR, 3’UTR, and poly(A) tail of variable length), but may be considerably longer (for example 9-12 kb).
  • the nucleic acid sequence preferably RNA, and most preferably saRNA, maybe at least 1000 bases in length, at least 2000 bases in length, at least 3000 bases in length, at least 4000 bases in length, at least 5000 bases in length, at least 6000 bases in length, at least 7000 bases in length, at least 8000 bases in length, at least 9000 bases in length at least 10000 bases in length, at least 11000 bases in length or at least 12000 bases in length.
  • the nucleic acid sequence is at least 6000 bases in length.
  • the RNA is at least 6000 bases in length.
  • the saRNA is at least 6000 bases in length.
  • the nucleic acid sequence, preferably RNA, and most preferably saRNA, may be between
  • the nucleic acid sequence is between 6000 and 15000 bases in length.
  • the nucleic acid sequence is between 8000 and 12000 bases in length.
  • the RNA is between 6000 and 15000 bases in length.
  • the RNA is between 8000 and 12000 bases in length.
  • the saRNA is between 6000 and 15000 bases in length.
  • the saRNA is between 8000 and 12000 bases in length.
  • nucleic acid is double stranded, for example double stranded RNA
  • bases in length will refer to the length of base pairs.
  • the RNA comprises or is derived from a positive stranded RNA virus selected from the group of genus consisting of: alphavirus; picornavirus;flavivirus; rubivirus; pestivirus; hepacivirus; calicivirus or coronavirus.
  • a positive stranded RNA virus selected from the group of genus consisting of: alphavirus; picornavirus;flavivirus; rubivirus; pestivirus; hepacivirus; calicivirus or coronavirus.
  • Suitable wild-type alphavirus sequences are well-known.
  • suitable alphaviruses include Aura, Bebaru virus, Cabassou, Chikungunya virus, Eastern equine encephalomyelitis virus, Fort Morgan, Getah virus, Kyzylagach, Mayaro, Mayaro virus, Middleburg, Mucambo virus, Ndumu, Pixuna virus, Ross River virus, Semliki Forest, Sindbis virus, Tonate, Triniti, Una, Venezuelan equine encephalomyelitis, Western equine encephalomyelitis, Whataroa and Y-62-33.
  • the RNA comprises or is derived from a virus selected from the group of species consisting of: Venezuelan Equine Encephalitis Virus (VEEV); enterovirus 71; Encephalomyocarditis virus; Kunjin virus; and Middle East respiratory syndrome virus.
  • VEEV Venezuelan Equine Encephalitis Virus
  • enterovirus 71 Encephalomyocarditis virus
  • Kunjin virus Kunjin virus
  • Middle East respiratory syndrome virus derived from VEEV.
  • the nucleic acid comprises a sequence which encodes the at least one therapeutic biomolecule.
  • the at least one therapeutic biomolecule may comprise or be a vaccine construct, or a therapeutic protein.
  • therapeutic protein relates to any protein that has therapeutic application, preferably in human.
  • Exemplary therapeutic biomolecules that can be encoded by the nucleic acid include proteins and peptides derived from pathogens, such as bacteria, viruses, fungi, protozoa/or parasites.
  • the protein and peptide is an antigen.
  • the protein and peptide derived from a virus maybe a viral antigen.
  • the viral antigen maybe derived from a virus selected from the group consisting of Orthomyxoviruses; Paramyxoviridae viruses; Metapneumovirus and Morbilliviruses; Pneumoviruses; Paramyxoviruses; Poxviridae; Metapneumoviruses; Morbilliviruses; Picomaviruses; Enteroviruseses; Bunyaviruses; Phlebovirus; Nairovirus; Hepamaviruses; Togaviruses; Alphavirus; Arterivirus; Flaviviruses; Pestiviruses; Hepadnaviruses; Rhabdoviruses; Caliciviridae Coronaviruses ; Retroviruses; Reoviruses; Parvoviruses; Delta hepatitis virus (HDV); Hepatitis E virus (HEV); Human Herpesviruses and Papova
  • the Orthomyxoviruses may be Influenza A, B and C.
  • Th eParamyxoviridae virus maybe Pneumoviruses (RSV), Paramyxoviruses (PIV).
  • the Metapneumovirus may be
  • Morbilliviruses e.g., measles.
  • the Pneumovirus maybe Respiratory syncytial virus (RSV), Bovine respiratory syncytial virus, Pneumonia virus of mice, or Turkey rhinotracheitis virus.
  • the Paramyxovirus maybe Parainkuenza virus types 1 - 4 (PIV), Mumps, Sendai viruses, Simian virus 5, Bovine parainkuenza virus, Nipahvirus, Henipavirus or Newcastle disease virus.
  • the Poxviridae may be Variola vera, for example
  • the Metapneumovirus maybe human metapneumovirus (hMPV) or avian metapneumoviruses (aMPV).
  • the Morbillivirus may be measles.
  • the Picornaviruses maybe Enteroviruses, Rhinoviruses, Hepamavirus, Parechovirus, Cardioviruses and Aphthoviruses.
  • the Enteroviruses maybe Poliovirus types 1, 2 or 3, Coxsackie A virus types 1 to 22 and 24, Coxsackie B virus types 1 to 6, Echovirus (ECHO) virus) types 1 to 9, 11 to 27 and 29 to 34 or Enterovirus 68 to 71.
  • the Bunyavirus may be California encephalitis virus.
  • the Phlebovirus may be Rift Valley Fever virus.
  • the Nairovirus maybe Crimean-Congo hemorrhagic fever virus.
  • the Hepamaviruses maybe Hepatitis A virus (HAV).
  • the Togaviruses maybe Rubivirus.
  • the Flavivirus may be Tick-borne encephalitis (TBE) virus, Dengue (types 1, 2, 3 or 4) virus, Yellow Fever virus, Japanese encephalitis virus, Kyasanur Forest Virus, West Nile encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus or Powassan encephalitis virus.
  • the Pestivirus may be Bovine viral diarrhea (BVDV), Classical swine fever (CSFV) or Border disease (BDV).
  • the Hepadnavirus maybe Hepatitis B virus or Hepatitis C virus.
  • the Rhabdovirus may be Lyssavirus (Rabies virus) or Vesiculovirus (VSV).
  • the Caliciviridae may be Norwalk virus, or Norwalk-like Viruses, such as Hawaii Virus and Snow Mountain Virus.
  • the Coronavirus maybe SARS, Human respiratory coronavirus, Avian infectious bronchitis (IBV), Mouse hepatitis virus (MHV), or Porcine transmissible gastroenteritis virus (TGEV).
  • the Retrovirus maybe Oncovirus, a Lentivirus or a Spumavirus.
  • the Reovirus may be an Orthoreo virus, a Rotavirus, an Orbivirus, or a Coltivirus.
  • the Parvovirus maybe Parvovirus B 19.
  • the Human Herpesvirus maybe Herpes Simplex Viruses (HSV), Varicella-zoster virus (VZV), Epstein- Barr virus (EBV), Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), Human Herpesvirus 7 (HHV7), or Human Herpesvirus 8 (HHV8).
  • the Papovavirus maybe Papilloma viruses , Polyomaviruses, Adenoviruess or Arenaviruses.
  • the protein and peptide derived from bacteria maybe a bacterial antigen.
  • the bacterial antigen may derived from a bacterium selected from the group consisting of: Neisseria meningitid.es, Streptococcus pneumoniae, Streptococcus pyogenes, Moraxella catarrhalis, Bordetella pertussis, Burkholderia sp.
  • Burkholderia mallei, Burkholderia pseudomallei and Burkholderia cepacia Staphylococcus aureus, Haemophilus inkuenzae, Clostridium tetani (Tetanus), Clostridium perfringens, Clostridium botulinums, Cornynebacterium diphtheriae (Diphtheria), Pseudomonas aeruginosa, Legionella pneumophila, Coxiella burnetii, Brucella sp. (e.g., B. abortus, B. canis, B. melitensis, B. neotomae, B.
  • ovis, B. suis and B. pinnipediaeJFrancisella sp. e.g., F. novicida, F. philomiragia and F. tularensis ), Streptococcus agalactiae, Neiserria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum (Syphilis), Haemophilus ducreyi, Enterococcus faecalis, Enterococcus faecium, Helicobacter pylori, Staphylococcus saprophyticus, Yersinia enter ocolitica, E.
  • F. novicida F. philomiragia and F. tularensis
  • Streptococcus agalactiae Neiserria gonorrhoeae
  • Chlamydia trachomatis Treponema pallid
  • coli Bacillus anthracis (anthrax), Yersinia pestis (plague), Mycobacterium tuberculosis, Rickettsia, Listeria , Chlamydia pneumoniae, Vibrio cholerae, Salmonella typhi (typhoid fever), Borrelia burgdorfer, Porphyromonas s and Klebsiella sp.
  • the protein and peptide derived from a fungus maybe a fungal antigen.
  • the fungal antigen maybe derived from a fungus selected from the group consisting of Dermatophytres, including: Epidermophyton koccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini, Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T verrucosum var.
  • Dermatophytres including: Epidermophyton koccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum g
  • album var. discoides, var. ochraceum, Trichophyton violaceum, and/or Trichophyton faviforme; or from Aspergillus fiimigatus, Aspergillus kavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus kavatus,
  • the protein and peptide derived from a protozoan maybe a protozoan antigen.
  • the protozoan antigen maybe derived from a protozoan selected from the group consisting of: Entamoeba histolytica, Giardia lambli, Cryptosporidium parvum,
  • Cyclospora cayatanensis Plasmodium species (vivax, ovale, malariae), Leishmania (donovani, tropica), Trypanosoma (brucei and cmzi) and Toxoplasma.
  • the protein and peptide derived from a helminth may be a helminth antigen.
  • the helminth antigen may be derived from a protozoan selected from the group consisting of: Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Strongyloides stercoralis andAncylostoma duodenale, Hymenolepis nana, Taenia saginata, Enterobius, Fasciola hepatica, Schistosoma mansoni, Toxocara canis and Toxocara cati
  • the therapeutic biomolecule maybe a protein and peptide derived from a plant.
  • the protein and peptide is a plant antigen.
  • the plant antigen maybe derived from Ricinus communis.
  • the antigen may be an allergen.
  • Allergens in this context include e.g. grasses, pollens, moulds, drugs, or numerous environmental triggers, etc. Allergy antigens typically belong to different classes of compounds, such as nucleic acids and their fragments, proteins or peptides and their fragments, carbohydrates, polysaccharides, sugars, lipids, phospholipids, etc.
  • the therapeutic biomolecule may be an immunogen or an antigen.
  • the immunogen or an antigen is a tumour immunogen or antigen, or cancer immunogen or antigen.
  • the tumour immunogens and antigens may be peptide-containing tumour antigens, such as a polypeptide tumour antigen or glycoprotein tumour antigens.
  • tumour antigens maybe (a) full length molecules associated with cancer cells, Ob) homologs and modified forms of the same, including molecules with deleted, added and/or substituted portions, and (c) fragments of the same.
  • Suitable tumour immunogens include: class I-restricted antigens recognized by CD 8+ lymphocytes or class II-restricted antigens recognized by CD4+ lymphocytes.
  • the tumour antigen maybe an antigen that is associated with a cancer selected from the group consisting of: a testis cancer, melanoma, lung cancer, head and neck cancer, NSCLC, breast cancer, gastrointestinal cancer, bladder cancer, colorectal cancer, pancreatic cancer, lymphoma, leukaemia, renal cancer, hepatoma, ovarian cancer, gastric cancer and prostate cancer.
  • the tumour antigen may be selected from:
  • cancer-testis antigens such as NY-ESO-I, SSX2, SCPl as well as RAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-I, GAGE-2, MAGE- 1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE- 12 (which can be used, for example, to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumours);
  • cancer-testis antigens such as NY-ESO-I, SSX2, SCPl as well as RAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-I, GAGE-2, MAGE- 1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE- 12 (which can be used, for example, to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumours);
  • mutated antigens for example, P53 (associated with various solid tumours, e.g., colorectal, lung, head and neck cancer), p2i/Ras (associated with, e.g., melanoma, pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g., melanoma), MUMl (associated with, e.g., melanoma), caspase-8 (associated with, e.g., head and neck cancer), CIA 0205 (associated with, e.g., bladder cancer), HLA-A2-R1701, beta catenin (associated with, e.g., melanoma), TCR (associated with, e.g., T- cell non-Hodgkins lymphoma), BCR- abl (associated with, e.g., chronic myelogenous leukemia), triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-
  • melanoma-melanocyte differentiation antigens such as MART-i/Melan A, gplOO, MC1R, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase related protein- 1 /TRPl and tyrosinase related protein-2/TRP2 (associated with, e.g., melanoma);
  • prostate-associated antigens such as PAP, PSA, PSMA, PSH-Pl, PSM-Pl, PSM-P2, associated with e.g., prostate cancer; and/ or
  • the therapeutic biomolecule may be a eukaryotic polypeptide.
  • the eukaryotic polypeptide is a mammalian polypeptide.
  • the mammalian polypeptide may be selected from the group consisting of: an enzyme; an enzyme inhibitor; a hormone; an immune system protein; a receptor; a binding protein; a transcription or translation factor; tumour growth supressing protein; a structural protein and a blood protein.
  • the enzyme maybe selected from the group consisting of: chymosin; gastric lipase; tissue plasminogen activator; streptokinase; a cholesterol biosynthetic or degradative steriodogenic enzyme; kinases; phosphodiesterases; methylases; de-methylases; dehydrogenases; cellulases; proteases; lipases; phospholipases; aromatases; cytochromes; adenylate or guanylaste cyclases and neuramidases.
  • the enzyme inhibitor maybe tissue inhibitor of metalloproteinase (TIMP).
  • the hormone may be growth hormone.
  • the immune system protein may be selected from the group consisting of: a cytokine; a chemokine; a lymphokine; erythropoietin; an integrin; addressin; selectin; homing receptors; T cell receptors and immunoglobulins.
  • the cytokine maybe an interleukin, for example IL-2, IL-4 and/or IL-6, colony stimulating factor (CSF), granulocyte colony stimulating factor (G- CSF), granulocyte- macrophage colony stimulating factor (GM-CSF) or tumour necrosis factor (TNF).
  • CSF colony stimulating factor
  • G- CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte- macrophage colony stimulating factor
  • TNF tumour necrosis factor
  • the chemokine may be a macrophage inflammatory protein-2 and/ or a plasminogen activator.
  • the lymphokine may be an interferon.
  • the immunoglobulin may be a natural, modified or chimeric immunoglobulin or a fragment thereof.
  • the immunoglobulin is a chimeric immunoglobulin having dual activity such as antibody enzyme or antibody-toxin chimera.
  • the hormone may be selected from the group consisting of: insulin, thyroid hormone, catecholamines, gonadotrophines, trophic hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptins; growth hormones (e.g., human grown hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor and the like).
  • the receptor maybe a steroid hormone receptor or a peptide receptor.
  • the receptor is a growth factor receptor.
  • the binding protein may be a growth factor binding protein.
  • the tumour growth suppressing protein maybe a protein that inhibits angiogenesis.
  • the structural protein may be selected from the group consisting of: collagen; fibroin; fibrinogen; elastin; tubulin; actin; and myosin.
  • the blood protein maybe selected from the group consisting of thrombin; serum albumin; Factor VII; Factor VIII; insulin; Factor IX; Factor X; tissue plasminogen activator; protein C; von Wilebrand factor; antithrombin III; glucocerebrosidase; erythropoietin granulocyte colony stimulating factor (GCSF) or modified Factor VIII; and anticoagulants.
  • GCSF erythropoietin granulocyte colony stimulating factor
  • the therapeutic biomolecule is a cytokine which is capable of regulating lymphoid homeostasis, preferably a cytokine which is involved in and preferably induces or enhances development, priming, expansion, differentiation and/or survival of T cells.
  • the cytokine is an interleukin. Most preferably, IL-2, IL-7, IL-12, IL-15, or IL-21.
  • the therapeutic biomolecule maybe protein that is capable of enhancing reprogramming of somatic cells to cells having stem cell characteristics.
  • the protein that is capable of enhancing reprogramming of somatic cells to cells having stem cell characteristics maybe selected from the group consisting of: OCT4, SOX2, NANOG, LIN28, p53, ART-4, BAGE, ss- catenin/m, Bcr-abL CAMEL, CAP-i, CASP-8, CDC27/m, CD 4/m, CEA, CLAUDIN-12, c- MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, GaplOO, HAGE, HER-2/neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A, MAGE-B, MAGE- C, MART- l/Melan- A, MC
  • MAGE-A is selected from the group consisting of: MAGE-A 1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE- A7, MAGE-A8, MAGE-A9, MAGE- A 10, MAGE-A 11, or MAGE-A 12
  • the protein that is capable of enhancing reprogramming of somatic cells to cells having stem cell characteristics is OCT4, SOX2, LF4; c-MYC; NANOG; LIN28.
  • the therapeutic biomolecule maybe a bio molecule that is utilised for the modification of cells ex vivo for cell-therapy indications.
  • the therapeutic biomolecule may be selected from the group consisting of an immunoglobulin, a T-cell receptor and NK receptor.
  • the therapeutic biomolecule maybe an RNA molecule that is capable of regulating expression of endogenous host genes, for example an interfering RNA such as small RNAs, siRNA or microRNAs.
  • the composition may comprise one or more additives. These additives maybe selected from a group consisting of: buffering substances; saccharides; stabilizers; cyroprotectants; lyoprotectants and chelating agents.
  • the compositions may be a frozen, lyophilized or sprayed dried composition.
  • the composition may comprise a cryoprotectant and/or lyoprotectant, for example a disaccharide such as trehalose, sucrose, maltose, or polysaccharide such as dextran or pullulan.
  • the compositions may further comprise a JAK/ STAT pathway inhibitor that is capable of disrupting potential innate responses to formulated nucleic acid, preferably saRNA.
  • the JAK/STAT pathway inhibitor maybe selected from a group consisting of: Ruxolitinib; Tofacitinib; Oclacitinib; Momelotinib; Baricitinib; Filgotinib; Itacitinib and similar inhibitors of JAK/STAT signalling that would be known to those skilled in the art.
  • the inventors believe comprising a polymeric composition and saRNA is novel and inventive per se.
  • RNA self-amplifying RNA
  • saRNA self-amplifying RNA
  • I formula (I):
  • L 1 to ⁇ J> are each independently an optionally substituted C - 2 alkylene, an optionally substituted C 2-i2 alkenylene, an optionally substituted C 2-12 alkynylene, an optionally substituted C 3-6 cycloalkylene, an optionally substituted 3 to 8 membered heterocyclylene, an optionally substituted Cc,- 12 arylene, an optionally substituted 5 to 10 membered heteroarylene or L 6 I7, wherein adjacent carbon atoms in the alkylene, alkenylene or alkynylene are optionally interrupted by one or more heteroatoms;
  • L 6 and 1 are independently an optionally substituted C - 2 alkylene, an optionally substituted C 2-i2 alkenylene, an optionally substituted C 2-i2 alkynylene, an optionally substituted C 3-6 cycloalkylene, an optionally substituted 3 to 8 membered heterocyclylene, an optionally substituted Ce- 12 arylene or an optionally substituted 5 to 10 membered heteroarylene, wherein adjacent carbon atoms in the alkylene, alkenylene or alkynylene are optionally interrupted by one or more heteroatoms;
  • R 1 and R 2 are each independently H, an optionally substituted CV 12 alkyl, an optionally substituted C 2-i2 alkenyl or an optionally substituted C 2-i2 alkynyl;
  • R3 is -OR 4 , -COOR 4 , -S0 2 0R4, (0CH 2 CH 2 ) m 0H, or NR 4 Rs,
  • R 4 and R 5 are each independently H, an optionally substituted C - i2 alkyl, an optionally substituted C 2-i2 alkenyl, an optionally substituted C 2-i2 alkynyl, an optionally substituted C 3-6 cycloalkyl, an optionally substituted 3 to 8 membered heterocyclyl, an optionally substituted C - 12 aryl or an optionally substituted 5 to 10 membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or alkenyl are optionally interrupted by one or more heteroatoms; and m is an integer between 1 and 10; or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof.
  • the average molecular mass of the plurality of polymers of formula (I) may be less than or equal to 5 kg mol ⁇ 1 . Notwithstanding the above, the plurality of polymers may be as defined in relation to the first aspect.
  • the saRNA maybe as defined in relation to the second aspect.
  • the weight ratio of polymeric composition and the saRNA maybe between 1:1 and 200:1, more preferably between 5:1 and 150:1 or between 10:1 and 100:1, and most preferably between 20:1 and 90:1, between 30:1 and 80:1, between 40:1 and 70:1 or between 45:1 and 60:1.
  • a nanoparticle comprising the composition of matter as defined in the second aspect or third aspect.
  • the inventors have found that the nanoparticle of the fourth aspect enhances transfection efficiency of nucleic acids in vitro.
  • the nanoparticle preferably has a hydrodynamic diameter (3 ⁇ 4) of less than 1,000 nm or less than 750 nm, more preferably less than 500 nm, less than 400 nm or less than 200 nm, and most preferably less than 150 nm, less than 100 nm, less than 80 nm or less than 75 nm.
  • the nanoparticle preferably has a hydrodynamic diameter (3 ⁇ 4) of between 1 and 1,000 nm or between 10 and 750 nm, more preferably between 20 and 500 nm, between 30 and 400 nm or between 40 and 200 nm, and most preferably between 50 and 150 nm, between 55 and 100 nm, between 60 and 80 nm or between 65 and 75 nm.
  • the hydrodynamic diameter may be calculated using dynamic light scattering (DLS).
  • the DLS measurements maybe taken when the nanoparticle is disposed in a buffer solution (20 Mm HEPES, 5 wt% glucose in water, pH 7.4), and measured using the Zetasizer Nano ZS instrument.
  • the scattering angle maybe fixed at 173 0 .
  • Data processing maybe carried out using cumulant analysis of the experimental correlation function and the Stokes-Einstein equation may be used to calculate the hydrodynamic radii.
  • a pharmaceutical composition comprising the polymeric composition of the first aspect, the composition of matter of the second or third aspect or the nanoparticle of the fourth aspect, and a pharmaceutically acceptable vehicle.
  • a vaccine comprising the polymeric composition of the first aspect, the composition of matter of the second or third aspect, the nanoparticle of the fourth aspect or the pharmaceutical composition of the fifth aspect.
  • the vaccine comprises a suitable adjuvant.
  • the adjuvant maybe an encoded molecular adjuvant that is encoded in the nucleic acid, as defined in relation to the second aspect, or as adjuvant incorporated into a delivery formulation.
  • the encoded molecular adjuvant may encode a cytokine, for example IL-2, IL-12, IL-21, GM-CSF, IFN-g, CCL20, CCL21, CXCL8, CXCLio, CXCL12 or an effector protein such as CD40L, Fit-3 or microbial protein, e.g flagellin or cholera toxin B.
  • the adjuvant incorporated into a delivery formulation may be selected from the group consisting of a bacterial lipopeptide, lipoprotein and lipoteichoic acid; mycobacterial lipoglycan; yeast zymosan, porin, Lipopolysaccharide, Lipid A, monophosphoryl lipid A (MPL), Flagellin, CpG DNA, hemozoin, Saponins (Quil-A, QS-21, Tomatine, ISCOM, ISCOMATRIXTM), squalene based emulsions, Carbopol, lipid nanoparticles and bacterial toxins (CT, LT).
  • a bacterial lipopeptide, lipoprotein and lipoteichoic acid may be selected from the group consisting of a bacterial lipopeptide, lipoprotein and lipoteichoic acid; mycobacterial lipoglycan; yeast zymosan, porin, Lipopolysaccharide, Lipid A, monophosphoryl lipid A (MPL
  • the immune response maybe stimulated against a helminth, protozoa, bacterium, virus, fungus or cancer as per the antigens defined in the second aspect.
  • the helminth, protozoan, fungal, bacterial or viral infection to be treated maybe an infection of a helminth, protozoa, fungus, bacterium or virus as defined in relation to the second aspect.
  • the cancer maybe as defined in relation to the second aspect.
  • a method for treating a helminth, protozoan, fungal, bacterial or viral infection comprising administering, or having administered, to a subject in need thereof, a therapeutically effective amount of the polymeric composition of the first aspect, the composition of matter of the second or third aspect, the nanoparticle of the fourth aspect, the pharmaceutical composition according to the fifth aspect, or the vaccine according to the sixth aspect.
  • the helminth, protozoan, fungal, bacterial or viral infection to be treated maybe an infection of a protozoa, fungus, bacterium or virus as defined in relation to the second aspect.
  • a method for treating cancer comprising administering, or having administered, to a subject in need thereof, a therapeutically effective amount of the polymeric composition of the first aspect, the composition of matter of the second or third aspect, the nanoparticle of the fourth aspect, the pharmaceutical composition according to the fifth aspect, or the vaccine according to the sixth aspect.
  • the cancer to be treated maybe as defined in relation to the second aspect.
  • Stem cell therapy may relate to the reprogramming somatic cells to cells having stem cell characteristics.
  • Somatic cells maybe reprogrammed by delivering one or more proteins that are capable of enhancing reprogramming of somatic cells to cells having stem cell characteristics as defined in relation to the second aspect.
  • a fourteenth aspect there is provided a method of modifying a cell ex vivo or in vitro, comprising delivering, to the cell, the polymeric composition of the first aspect, the composition of matter of the second or third aspect, the nanoparticle of the fourth aspect or the pharmaceutical composition according to the fifth aspect.
  • the method is performed ex vivo.
  • the method is performed in vitro.
  • the cell maybe a eukaryotic or prokaryotic cell.
  • the cell is a eukaryotic cell. More preferably, the cell is a mammalian host cell. Most preferably the cell is a human cell.
  • the modified cell is suitable for cell-therapy indications.
  • a modified cell obtained from, or obtainable by, the method of the fourteenth aspect.
  • the modified cell of the fifteenth aspect for use in therapy, optionally cell therapy.
  • the polymeric composition of the first aspect, the composition of matter of the second or third aspect, the nanoparticle of the fourth aspect, the pharmaceutical composition according to the fifth aspect, or the vaccine according to the sixth aspect may be used in a medicament, which maybe used as a monotherapy (i.e. use of the active agent), for treating, ameliorating, or preventing disease or vaccination.
  • the active agents according to the invention maybe used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing disease.
  • compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used.
  • the composition maybe in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension, polyplex, emulsion, lipid nanoparticles (with RNA on the surface or encapsulated) or any other suitable form that may be administered to a person or animal in need of treatment or vaccination.
  • vehicle of medicaments according to the invention should be one which is well-tolerated by the subject to whom it is given.
  • the polymeric composition, nanoparticle, pharmaceutical composition, or vaccine of the invention may also be incorporated within a slow- or delayed-release device.
  • a slow- or delayed-release device Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months.
  • the device maybe located at least adjacent the treatment site. Such devices maybe particularly advantageous when long-term treatment with the polymeric composition, nanoparticle, pharmaceutical composition, or vaccine is required and which would normally require frequent administration (e.g. at least daily injection).
  • medicaments according to the invention may be administered to a subject by injection into the blood stream, muscle, skin or directly into a site requiring treatment.
  • Injections maybe intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion), or intramuscular (bolus or infusion).
  • amount of polymeric composition, nanoparticle, pharmaceutical composition, or vaccine that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the polymeric composition, nanoparticle, pharmaceutical composition, or vaccine and whether it is being used as a monotherapy or in a combined therapy.
  • the frequency of administration will also be influenced by the half- life of the active agent within the subject being treated.
  • Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular polymeric composition, nanoparticle, pharmaceutical composition, or vaccine in use, the strength of the pharmaceutical composition, the mode of administration, and the type and advancement of the viral infection. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, sex, diet and time of administration.
  • a daily dose of between o.ooiug/kg of body weight and lomg/kg of body weight, or between o.oipg/kg ofbody weight and lmg/kg of body weight, of the polymeric composition, nanoparticle, pharmaceutical composition, or vaccine of the invention may be used for treating, ameliorating, or preventing a disease, depending upon the active agent used.
  • Daily doses maybe given as a single administration (e.g. a single daily injection or inhalation of a nasal spray).
  • the polymeric composition, nanoparticle, pharmaceutical composition, or vaccine may require administration twice or more times during a day.
  • the polymeric composition, nanoparticle, pharmaceutical composition, or vaccine may be administered as two (or more depending upon the severity of the disease being treated) daily doses of between 0.07 pg and 700 mg (i.e. assuming a body weight of 70 kg).
  • a patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter.
  • a slow release device maybe used to provide optimal doses of the polymeric composition, nanoparticle, pharmaceutical composition, or vaccine according to the invention to a patient without the need to administer repeated doses.
  • the polymeric composition, nanoparticle, pharmaceutical composition, or vaccine according to the invention maybe given as a weekly dose, and more preferably a fortnightly dose.
  • the polymeric composition, nanoparticle, pharmaceutical composition, or vaccine may be given as a single dose.
  • compositions and medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or maybe used in other veterinary applications. Most preferably, however, the subject is a human being.
  • a “therapeutically effective amount” of the polymeric composition, nanoparticle, pharmaceutical composition, or vaccine is any amount which, when administered to a subject, is the amount of the aforementioned that is needed to ameliorate, prevent or treat any given disease.
  • the polymeric composition, nanoparticle, pharmaceutical composition, or vaccine of the invention maybe used maybe from about o.oooi mg to about 800 mg, and preferably from about 0.001 mg to about 500 mg. It is preferred that the amount of polymeric composition, nanoparticle, pharmaceutical composition, or vaccine is an amount from about 0.01 mg to about 250 mg, and most preferably from about 0.01 mg to about 1 mg.
  • the polymeric composition, nanoparticle, pharmaceutical composition, or vaccine according to the invention is administered at a dose of 1-
  • a “pharmaceutically acceptable vehicle” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.
  • the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet.
  • a solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet- disintegrating agents.
  • the vehicle may also be an encapsulating material.
  • the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention.
  • the active agent e.g. polymeric composition, nanoparticle, pharmaceutical composition, or vaccine according to the invention
  • a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired.
  • the powders and tablets preferably contain up to 99% of the active agents.
  • Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
  • the pharmaceutical vehicle maybe a gel and the composition may be in the form of a cream or the like.
  • the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution.
  • Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
  • the polymeric composition, nanoparticle, pharmaceutical composition, or vaccine according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
  • the liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators.
  • liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil).
  • the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate.
  • Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration.
  • the liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.
  • Liquid pharmaceutical compositions which are sterile solutions or suspensions, can be utilized by, for example, subcutaneous, intradermal, intrathecal, epidural, intraperitoneal, intravenous and particularly intramuscular injection.
  • the polymeric composition, nanoparticle, pharmaceutical composition, or vaccine of the invention maybe prepared as a sterile solid composition that maybe dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.
  • the polymeric composition, nanoparticle, pharmaceutical composition, or vaccine of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 8o (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
  • the polymeric composition, nanoparticle, pharmaceutical composition, or vaccine according to the invention can also be administered orally either in liquid or solid composition form.
  • compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions.
  • forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
  • a method of producing a high molar mass polyfamido amine comprising contacting a compound of formula (II): with a compound of formula (III):
  • R and R are each independently H, an optionally substituted C - 12 alkyl, an optionally substituted C 2-12 alkenyl or an optionally substituted C 2-12 alkynyl;
  • R 6 to R 11 are each independently H, a halogen, an optionally substituted C - 12 alkyl, an optionally substituted C 2-12 alkenyl, an optionally substituted C 2-12 alkynyl, an optionally substituted C 3-6 cycloalkyl, an optionally substituted 3 to 8 membered heterocyclyl, an optionally substituted C 6-12 aryl or an optionally substituted 5 to 10 membered heteroaryl;
  • L 2 and I are each independently absent or an optionally substituted C - 12 alkyl ene, an optionally substituted C 2-12 alkenylene, an optionally substituted C 2-12 alkynylene, an optionally substituted C 3-6 cycloalkylene, an optionally substituted 3 to 8 membered heterocyclylene, an optionally substituted C 6-12 arylene, an optionally substituted 5 to 10 membered heteroarylene or L 6 L 7 , wherein adjacent carbon atoms in the alkylene, alkenylene or alkyny
  • R 12 is an optionally substituted C - 2 alkyl, an optionally substituted C 2-12 alkenyl, an optionally substituted C 2-12 alkynyl, an optionally substituted C 3 -6 cycloalkyl, an optionally substituted 3 to 8 membered heterocyclyl, an optionally substituted C 6-12 aryl, an optionally substituted 5 to 10 membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or alkenyl are optionally interrupted by one or more heteroatoms; and R 13 is H, an optionally substituted C - 12 alkyl, an optionally substituted C 2-12 alkenyl, an optionally substituted C 2-12 alkynyl, an optionally substituted C 3 -6 cycloalkyl, an optionally substituted 3 to 8 membered heterocyclyl, an optionally substituted C 6-12 aryl, an optionally substituted 5 to 10 membered heteroaryl, wherein adjacent carbon atoms in the alkyl,
  • the method of the seventeenth aspect may be used to produce a compound according to the first aspect.
  • R 1 and R 2 are as defined in relation to the first aspect.
  • R 6 to R 11 are each independently H, a halogen, an optionally substituted C - 3 alkyl, an optionally substituted C 2-3 alkenyl or an optionally substituted C 2-3 alkenyl. More preferably, R 6 to R 11 are each H.
  • L 2 and L 3 are as defined in relation to the first aspect.
  • L 8 is -S-S-.
  • the compound of formula (II) maybe a compound of formula (Ila): Accordingly, the compound of formula (II) may be N,N’-cystaminebisacrylamide (CBA).
  • R 12 is an optionally substituted C 2- s alkyl, an optionally substituted C 2- s alkenyl or an optionally substituted C 2- s alkynyl. More preferably R 12 is an optionally substituted C3-5 alkyl, an optionally substituted C 3-5 alkenyl or an optionally substituted C 3-5 alkynyl.
  • the alkyl, alkenyl or alkynyl is preferably substituted.
  • the alkyl, alkenyl or alkynyl is substituted with an OR 14 group.
  • R 14 is H.
  • R 13 is H, an optionally substituted C - 6 alkyl, an optionally substituted C 2-6 alkenyl or an optionally substituted C 2-6 alkynyl. More preferably, R 13 is H, an optionally substituted C - 3 alkyl, an optionally substituted C 2-3 alkenyl or an optionally substituted C 2- 3 alkynyl. Most preferably, R 13 is H.
  • the compound of formula (III) maybe 4-amino-i-butanol (ABOL).
  • the Lewis base maybe ammonia or an amine.
  • the amine maybe a primary amine, a secondary amine or a tertiary amine.
  • the tertiary amine may be trimethyl amine or triethyl amine.
  • the Lewis base is triethyl amine (TEA).
  • the molar ratio of the compound of formula (II) to the compound of formula (III) is preferably between 1:1 and 1,000:1, more preferably is between 2:1 and 100:1, between 3:1 and 75:1 or between 4:1 and 50:1, and most preferably is between 5:1 and 25:1, between 6:1 and 20:1, between 7:1 and 15:1, between 8:1 and 12:1 or between 9:1 and 11:1. In a preferred embodiment, the molar ratio of the compound of formula (II) to the compound of formula (III) is about 10:1.
  • the molar ratio of the compound of formula (II) to the Lewis base is preferably between 1:1 and 1,0000:1, more preferably is between 10:1 and 1,000:1, between 25:1 and 750:1 or between 50:1 and 500:1, and most preferably is between 60:1 and 250:1, between 70:1 and 200:1, between 80:1 and 150:1, between 90:1 and 120:1 or between 95:1 and 110:1.
  • the molar ratio of the compound of formula (II) to the Lewis base is about 100:1.
  • the molar ratio of the compound of formula (III) to the Lewis base is preferably between 1:1 and 1,000:1, more preferably is between 2:1 and 100:1, between 3:1 and 75:1 or between 4:1 and 50:1, and most preferably is between 5:1 and 25:1, between 6:1 and 20:1, between 7:1 and 15:1, between 8:1 and 12:1 or between 9:1 and 11:1.
  • the molar ratio of the compound of form formula (III) to the Lewis base is about 10:1.
  • the compounds of formula (II) and (III) maybe contacted in a first solvent, to create a reactive solution.
  • the first solvent may comprise an alcohol and/or water.
  • the alcohol may be a C - 6 alcohol, and is preferably methanol or ethanol, and most preferably is methanol.
  • the first solvent comprises an alcohol and water.
  • the volumetric ratio of the alcohol to water maybe between 1:5 and 50:1, more preferably between 1:2 and 25:1 or between 1:1 and 10:1, and most preferably is between 2:1 and 8:1 or between 3:1 and 5:1. In a preferred embodiment, the ratio of alcohol to water is about 4:1.
  • the concentration of the compound of formula (II) in the solvent maybe between 0.1 and 50 M, more preferably is between 0.5 and 25 M or between 1 and 10 M, and most preferably is between 2 and 8M or between 4 and 6 M.
  • the concentration of the compound of formula (III) in the solvent maybe between 0.01 and 5 M, more preferably is between 0.05 and 2.5 M or between 0.1 and 1 M, and most preferably is between 0.2 and 0.8 M or between 0.4 and 0.6 M.
  • the concentration of the Lewis base in the solvent may be between 0.001 and 0.5 M, more preferably is between 0.005 and 0.25 M or between 0.01 and 0.1 M, and most preferably is between 0.02 and 0.08 M or between 0.04 and 0.06 M.
  • the compounds of formula (II) and (III) maybe contacted at a temperature of between o and ioo°C, more preferably between 5 and 90°C, between 10 and 8o°C or between 20 and 70°C, and most preferably between 30 and 6o°C or between 40 and 50°C.
  • the compounds of formula (II) and (III) maybe contacted in the dark.
  • the compounds of formula (II) and (III) may be contacted for between 1 hour and 50 days, more preferably between 1 and 25 days or between 2 and 20 days, and most preferably between 3 and 18 days, between 4 and 15 days or between 5 and 14 days. It may be appreciated that the exact amount of time may vary depending upon the desired molecular mass of the polymer.
  • the method may comprise stopping the reaction.
  • the method may comprise stopping the reaction by quenching the reaction solution with a second solvent.
  • the second solvent may be an alcohol or water.
  • the alcohol may be a C - 6 alcohol, and is preferably methanol or ethanol, and most preferably is methanol.
  • the volumetric ratio of the first solvent to the second solvent maybe between 1:1 and 1:10,000, more preferably between 1:10 and 1:1,000 or between 1:50 and 1:750, and most preferably between 1:100 and 1:500, between 1:200 and 1:400 or between 1:250 and 1:300.
  • the method may comprise contacting the high molar mass poly(amido amine) with an acid.
  • the acid maybe any inorganic or organic acid, such as hydrochloric acid, sulfuric acid, nitric acid or acetic acid. .
  • the acid maybe added at a concentration of between 0.01 and 100 M, more preferably between 0.05 and 50 M or between 0.1 and 10 M, and most preferably between 0.2 and 7.5 M, between 0.5 and 5 M, between 0.75 and 2.5 M or between 0.9 and 1.5 M.
  • the method may comprise purifying the high molar mass poly(amido amine).
  • the high molar mass poly(amido amine) maybe purified using dialysis, preferably against acidic water.
  • a method of producing a composition of matter or a nanoparticle comprising contacting a polymeric composition as defined in the first aspect with a nucleic acid.
  • the method may comprise producing the composition of matter of the second or third aspect or the nanoparticle of the fourth aspect.
  • the nucleic acid maybe as defined in relation to the second aspect.
  • the weight ratio of polymeric composition to the nucleic acid maybe between 1:1 and 200:1, more preferably between 5:1 and 150:1 or between 10:1 and 100:1, and most preferably between 20:1 and 90:1, between 30:1 and 80:1, between 40:1 and 70:1 or between 45:1 and 60:1.
  • the method may comprise contacting a first solution, comprising the polymeric composition, with a second solution, comprising the nucleic acid.
  • the first solution may comprise a solvent.
  • the solvent may comprise water and/or a buffer.
  • the solvent comprises water and a buffer.
  • the buffer maybe configured to maintain a pH between 5 and 9 at 20°C, more preferably a pH of between 6 and 8 or between 7 and 7.8 at 20°C, and most preferably a pH between 7.2 and
  • the buffer may be a HEPES buffer.
  • the first solution may comprise the polymeric composition at a concentration of between 0.0001 and 500 pg/pL, more preferably between 0.001 and 100 pg/pL or between 0.01 and 50 pg/ pL, and most preferably between 0.05 and 10 pg/ pL, between 0.1 and 1 pg/ pL or between 0.2 and 0.5 pg/ pL.
  • the second solution may comprise a solvent.
  • the solvent may comprise water and/or a buffer.
  • the solvent comprises water and a buffer.
  • the buffer may be configured to maintain a pH between 5 and 9 at 20°C, more preferably a pH of between 6 and 8 or between 7 and 7.8 at 20°C, and most preferably a pH between 7.2 and 7.6 at 20°C.
  • the buffer may be a HEPES buffer.
  • the second solution may comprise the nucleic acid at a concentration of between 0.00001 and 10 pg/ pL, more preferably between 0.0001 and 1 pg/ pL or between 0.0005 and 0.5 pg/pL, and most preferably between 0.0008 and 0.05 pg/pL or between 0.001 and 0.005 Pg/pL-
  • the volumetric ratio of the first solution to the second solution maybe between 10:1 and 1:20, more preferably between 2:1 and 1:10 or between 1:1 and 1:8, and most preferably is between 1:2 or 1:6 or between 1:3 and 1:5.
  • the second solution may be added to the first solution over a period of time.
  • the first solution maybe stirred constantly as the second solution is added thereto.
  • the second solution may be added to the first solution at an approximately constant rate.
  • the period of time may be between 1 second and 24 hours, more preferably between 10 seconds and 1 hour or between 30 seconds and 30 minutes, and most preferably is between 1 and 20 minutes, 2 and 10 minutes, 3 and 8 minutes or 4 and 6 minutes.
  • the inventors were able to obtain nanoparticles with diameters of about 70 nm.
  • a process for making the pharmaceutical composition according to the fifth aspect comprising contacting the polymeric composition of the first aspect, the composition of matter of the second or third aspect or the nanoparticle of the fourth aspect, with a pharmaceutically acceptable vehicle.
  • Figure 1 is a schematic illustration of (a) improved Aza-Michael addition to afford high molar mass poly(amido amine)s, poly(CBA-4-amino-i-butanol)s pABOLs with molar masses up to 167 kg mol ⁇ 1 ; (b) complexation with self-amplifying RNA (saRNA) via titration method and transfection efficacy of the pABOL-100 polypi exes, comparing to jetPEI andPEI MAX;
  • saRNA self-amplifying RNA
  • Figure 2 shows the synthesis of high MW pABOL and characterization of resulting saRNA polyplexes.
  • mice were injected with 5 pg of saRNA in each leg, and a ratio polymer to RNA of 45:1 (w/w) for pABOL.
  • Each circle represents one leg of one animal, and bar represents mean +/- SEM.
  • Figure 5 shows the effect of molecular weight, route of administration and ratio of pABOL to RNA on in vivo expression of fLuciferase-encoding saRNA polyplexes.
  • a-b Quantification of fLuc expression of PEI (jetPEI and PEI MAX) and pABOL polyplexes in total flux (p/s), 7 d after injection.
  • mice were injected with 5 pg of saRNA either (a) intramusculary or (b) intradermally, and a ratio polymer to RNA of 45:1 (w/w) for pABOL, 1:1 for PEI MAX, and an N:P of 8 for jetPEI.
  • Each circle represents one leg of one animal, and bar represents mean +/- SD.
  • Each circle represents one leg of one animal, and bar represents mean +/- SD.
  • f Representative images of each group, corresponding to e).
  • d-f Histograms of mean eGFP fluorescence intensity (MFI) for each formulation in human skin explants (d), IM injection in mice (e) and ID injection in mice (f).
  • MFI mean eGFP fluorescence intensity
  • e IM injection in mice
  • f ID injection in mice
  • Figure 7 shows phenotypic identity of cell present in human skin explants and GFP+ cells after intradermal (ID) injection of polyplex formulations as determined by flow cytometry a) Identity of cells in the population of total cells extracted from human skin explants; and b) identity of GFP-expressing skin cells from explants treated with polyplex formulated eGFP-encoding saRNA.
  • FIG. 8 shows immunogenicity of HA-encoding saRNA polyplexes.
  • a-b Change in body weight after IN challenge with Cal/09 flu virus for mice injected either IM (a) or ID (b).
  • Figure 9 shows graphs monitoring the (a) molecular weight and (b) molecular weight dispersity (£>) over time using SEC, corresponding to 1 M (A), 5 M ( ⁇ ) and 5 M with TEA ( ⁇ ).
  • Poly(amido amine)s fit the inventors’ polymeric criteria and in addition, depending on the monomer combinations, linear pAAs generally have good water solubility, stability against hydrolysis and tunable degradation.
  • a disulphide monomer N,N’-cystaminebisacrylamide (CBA)
  • CBA disulphide monomer
  • GSH glutathione
  • preparation of pAAs is simple; two monomers are mixed together and undergo Aza-Michael polyaddition, which is a facile approach for scale-up and clinical translation.
  • previous reports on pAAs reports polymers with molar mass limited to ⁇ 5 kg mol ⁇ 1 with ⁇ io repeat units, 16-19 which to be more accurate, are just oligomers.
  • the inventors prepared a library of poly(CBA-4-amino-i-butanol) (pABOL) (see Figure l) with varying molar mass, ranging from 5 to 167 kg mol -1 , using an optimized Aza-Michael polyaddition synthesis protocol.
  • pABOL poly(CBA-4-amino-i-butanol)
  • Figure l poly(CBA-4-amino-i-butanol)
  • the inventors characterized the relationship between pABOL molar mass and protein expression in vivo using both intramuscular (IM) and intradermal (ID) injection. They then assessed whether protein expression was due to the quality or quantity of cellular expression ex vivo in human skin explants and in vivo in murine skin and muscle and phenotyped the cells in human skin that express pABOL/saRNA complexes. Finally, the inventors use pABOL and hemagglutinin (HA)- encoding saRNA as a vaccine model and observe the immunogenicity and ability to protect against influenza challenge compared to pEI in vivo.
  • IM intramuscular
  • ID intradermal
  • the targeted conversions can be easily achieved within 3 days.
  • the conversions were not monitored after 4 days as the double bond conversion exceeded 99.9% in the catalysed reaction; thus the residual signals were too weak to be detected in the NMR spectroscopy even with 1024 scans.
  • higher molar masses are accessible by extending reaction period from 5 to 14 days.
  • pABOLs with molar masses ranging from 5 to 167 kg mol ⁇ 1 (Table 1) were successfully prepared via the improved aza- Michael polyaddition conditions.
  • the inventors were able to synthesize pABOLs with molar masses > 30 kg mol ⁇ 1 .
  • the method described here may enable synthesis of high molar mass poly(amido amine)s given the broad range of commercial chemicals that undergo aza-Michael polyaddition.
  • the polyplexes were prepared via a direct mixing procedure. Given that the binding sites on both the saRNA and high molar mass pABOLs might not be completely accessible, due to the higher-order structure and the sterically hindered tertiary amine groups, respectively, the inventors opted to use a range of polymer/RNA weight ratios (from i:i to 6o:i) instead of the commonly used N /P values. It is noteworthy that theoretical average molar masses per charge of pABOLs and saRNA are 349.5 g mol 1 and 339.5 g mol ⁇ 1 , respectively, suggesting the weight ratios are close to N /P values.
  • PEI firefly luciferase
  • the inventors also tested whether increasing the molar mass of pABOL similarly enhanced the transfection efficiency mRNA and plasmid DNA (pDNA). Although the enhancement in mRNA and pDNA transfection was not as significant as in saRNA, it implies the molar mass effect is not only specifically applied to long-chain nucleic acids, like saRNA, but to other nucleic acid species as well. This knowledge is useful for the future design of polymer-based delivery systems for nucleic acids. In addition to transfection efficiency, the inventors also evaluated the in vitro cytotoxicity of saRNA/pABOL formulations (Figure 3c). Compared to PEI, pABOLs display less cytotoxicity. Furthermore, a molar mass dependence was observed for pABOLs.
  • pABOLs with low/moderate molar masses (8 and 25 kg mol 1 ) demonstrate much less cytotoxicity, comparing to their high molar mass analogues (72 and too kg mol 1 ), which could be due to surface charge and/ or the concentration of free polycations.
  • the inventors then sought to determine the role of bioreduction of pABOLs on in vitro transfection efficiency. As a bioreducible polycation, it is hypothesized that pABOL releases saRNA via the intracellular glutathione (GSH) reduction of the disulfide bonds on its backbone. 16 To confirm that pABOL is capable of being reduced by GSH, the bioreduction of pABOLs was monitored using GSH and the reduced product was identified to be a dithiol compound. The inventors then used a known GSH inhibitor, buthionine sulphoximine (BSO), 30 to pretreat cells and evaluate whether pABOLs had the same transfection with normal or reduced intracellular levels of GSH.
  • BSO buthionine sulphoximine
  • saRNA-polyplexes that can undergo filter sterilization (0.2 pm) without loss of activity.
  • the inventors optimized a titration method to prepare polyplexes with a size of ⁇ 100 nm. Titrating saRNA solutions (800 pL, 1.00 x to -3 mg mL 1 ) into polymer solutions (200 pL, 0.18 mg mL ⁇ 1 ) at a flow rate of 160 pL min 1 yields smaller nanoparticles with a hydrodynamic diameter of ⁇ 70 nm, narrow dispersity (0.2) and high surface charge (+ 23 mV) ( Figure 4a).
  • Example 4 Increasing pABOL molar mass enhances luciferase expression in vivo
  • the inventors further investigated whether increasing the molar mass of pABOLs enhanced the delivery and expression of saRNA in vivo, using fLuc as a reporter protein (Figure 5). They tested a range of pABOL molecular weights, from 8 to 167 kg mol 1 , as these were the polymers that they found to effectively complex and condense saRNA ( Figure 2b and 2c).
  • mice were injected with 5 pg of fLuc saRNA/pABOL polyplexes prepared at ratio of 45:1 (w/w) either intramuscularly (IM) or intradermally (ID) and imaged after 7 d, which has been previously shown to be peak protein expression for Venezuelan equine encephalitis virus (VEEV). ⁇
  • the inventors used two commercially available linear PEIs as a positive control — PEI MAX, which was used in all of their transfection experiments, and in vivo jetPEI®, which has previously been show to more effectively deliver RNA in vivo.
  • the inventors postulate that this parabolic relationship is governed by a mechanism wherein high luciferase expression from the 8 kg mol 1 pABOL polyplexes results from more rapid reduction and thus rapid uptake of RNA in vivo, whereas the higher molar mass polymers are reduced less quickly but provide more adequate protection for the RNA potentially resulting high intracellular RNA delivery.
  • the pABOLs with moderate molar mass 25 and 41 kg mol ⁇ 1
  • the inventors sought to determine the optimal ratio of pABOL to saRNA in vivo ( Figure 5e,f).
  • Example 5 - pABOL enhances the quantity of cells expressing saRNA both in vivo and ex vivo in human skin explants
  • the inventors then sought to investigate whether pABOLs enhance the quality or quantity of cells expressing saRNA both ex vivo in a clinically relevant human skin explant model and in vivo in mouse muscle and skin.
  • the inventors compared saRNA alone, the commercially available PEIs (PEI MAX and jetPEI) and 25, 72 and too kg mol ⁇ 1 pABOL complexed with 2 pg of enhanced green fluorescent protein (eGFP) saRNA ( Figure 6 a,d).
  • PEI MAX and jetPEI the commercially available PEIs
  • eGFP enhanced green fluorescent protein
  • RNA alone resulted in eGFP expression in ⁇ i% of human skin cells ( Figure 6a), and complexation with PEI MAX and jetPEI did not increase the number of eGFP- positive cells.
  • RNA alone had an eGFP MFI of ⁇ io 2 , and none of the formulations enhanced the protein expression per cell, which would manifest as a shift to the right on the x-axis of the histogram plot in Figure 6d. It is hypothesized that this is due to the self-replicating nature of the VEEV vector, wherein upon entering a cell it exhausts the cellular translational machinery resulting in the maximum MFI per cell.
  • MFI median fluorescence intensity
  • RNA alone yielded expression in ⁇ io% of cells when injected IM, which was only enhanced to ⁇ 20% of cells by 41 and too kg mol 1 pABOL (p 0.0074 and 0.0022, respectively). 8 kg mol ⁇ 1 pABOL enhanced the eGFP + cells to 16%, but this was not statistically significant.
  • RNA alone yielded eGFP expression in 20% of cells after ID injection which was increased to ⁇ 30% of cells with 8 and too kg mol ⁇ 1 pABOL, although they were not statistically significant.
  • jetPEI and 41 kg mol ⁇ 1 pABOL did not enhance the number of eGFP + cells when injected ID.
  • the inventors postulate that this is due to differential cell types between the muscle and the skin, which may have different kinetics of pABOL reduction and saRNA expression. Similar to the human skin explants, there was no significant shift in GFP MFI (Figure 6e,f), further indicating that the total protein expression relies on the number of cells and not the amount of protein being expressed by each cell.
  • pABOL enhances the percentage of cells expressing saRNA compared to RNA alone or commercially available PEIs, when injected IM or ID in vivo in mice or ID ex vivo in human skin explants.
  • Example 6 - pABOL-delivered saRNA is preferentially expressed bv epithelial cells in human skin explants
  • the inventors then further investigated which cells in human skin explants were expressing eGFP saRNA after intradermal injection (Figure 7).
  • the inventors observed that human skin explants are composed primarily of epithelial cells (53.7%), dendritic cells (14.8%), fibroblasts (11.6%) and Langerhans cells (10.8%) (Figure 7a).
  • the remaining 9% is composed of more rare immune cells, including leukocytes (4.0%), natural killer (NK) cells (2.6%), T cells (1.7%), B cells (0.6%) and monocytes (0.2%).
  • RNA alone and PEIs were the dominant cell type expressing the saRNA (18-24%), followed by DCs, leukocytes, Langerhans cells, B cells, fibroblasts, monocytes, NK cells and T cells.
  • the inventors hypothesize that the predominant uptake of RNA alone and PEIs by mostly immune cells indicates that these formulations are scavenged by professional immune cells, whereas the pABOL formulations may actively enhance cellular uptake into epithelial cells.
  • Example 7 Hemagluttinin (HA) saRNA/ pABOL polyplexes induce high HA antibody titers and confer complete protection against flu challenge in vivo
  • mice receive a prime and boost of either 1 or 0.1 pg of saRNA complexed with either jetPEI, 8 kg mol ⁇ 1 pABOL or too kg mol ⁇ 1 pABOL at a ratio of 45:1 (w/w).
  • the boost was administered 6 weeks after the initial prime.
  • the mice were challenged IN with Cal/09 flu virus three weeks after the boost and weighed daily to monitor disease progression.
  • mice in both the IM and ID groups all lost >25% of their body weight between days 4-6 and had to be culled according to the challenge protocol ( Figure 8a, b).
  • the IM injection groups all mice in the PEI and 8 kg mol ⁇ 1 pABOL groups were completely protected, even in the 0.1 pg groups, with the 1 pg 8 kg mol ⁇ 1 pABOL group showing the least amount of weight loss at peak viremia ( ⁇ 8%). All the mice in the 1 pg too kg mol ⁇ 1 pABOL group were completely protected, but two mice in the 0.1 pg too kg mol ⁇ 1 pABOL group reached 25% weight loss on day 5 and had to be culled, thus resulting in 60% survival in this group.
  • the HA IgG antibody titers (Figure 8b) directly reflect the challenge results; all groups show increasing antibody titers between 3 and 6 weeks, and then after the boost.
  • the 1 pg 8 kDa pABOL group had the highest antibody titers ( ⁇ 40,000 ng/mL) after 9 weeks, whereas the PEI and too kg mol-i pABOL groups that received 1 pg were equivalent ( ⁇ io,ooo ng/mL).
  • the ID injection groups were less protective against influenza challenge. Only the 1 pg PEI group conferred complete protection, and resulted in ⁇ 12% weight loss during peak viremia.
  • the 1 pg 8 kg mol ⁇ 1 pABOL had ⁇ 20% weight loss after 5 days and only reached antibody titers of ⁇ 500 ng/ mL.
  • the 0.1 pg PEI, 0.1 pg 8 kg mol ⁇ 1 pABOL and 1/0.1 pg 100 kg mol ⁇ 1 pABOL groups all had approximately equivalent antibody titers, never reaching more than ⁇ ioo ng/mL and exhibiting low survival.
  • NMR ⁇ , «G ⁇ H ⁇ , ⁇ - ⁇ COSY and HSQC NMR spectra were recorded using a Bruker AV 400 MHz spectrometer at room temperature.
  • DLS Dynamic light scattering was used to determine the hydrodynamic diameter (Du) and polydispersity of the nanostructures formed between PABOLs and saRNA, in buffer solutions (20 Mm HEPES, 5 wt% glucose in water, pH 7.4), and was measured using the Zetasizer Nano ZS instrument. The scattering angle was fixed at 173 0 .
  • Data processing was carried out using cumulant analysis of the experimental correlation function and the Stokes-Einstein equation was used to calculate the hydrodynamic radii. All solutions were analyzed using disposable polystyrene cuvettes.
  • Zeta potential measurements were also conducted at 25 °C using a ZETASIZER Nano ZS instrument.
  • Nanodrop The saRNA recovery was monitored using a Nanodrop One (Thermo Fisher) before and after sterile filtration of the polyplexes, through a 0.2 pm syringe filter (membrane material: hydrophilic PVDF). Mass spectroscopy: Mass spec characterizations were conducted using a Waters LCT Premier Mass Spectrometer. Samples were ionized using the electrospray (ES) technique.
  • ES electrospray
  • TEM Transmission electron microscopy
  • PABOL was synthesized by aza-Michael polyaddition of 4-amino-i-butanol (ABOL) to N,N’-cystaminebisacrylamide (CBA).
  • CBA 4-amino-i-butanol
  • CBA N,N’-cystaminebisacrylamide
  • CBA 4-amino-i-butanol
  • ABOL 78 pL, 0.840 mmol
  • trimethylamine (12 pL, 0.084 mmol
  • the mixture was allowed to react for 5 to 14 days (depending on the targeted molecular weight) to yield a highly viscous solution. Aliquots were taken at predetermined time intervals for ⁇ NMR and SEC to monitor the conversion and molar mass. The reaction was stopped by MeOH dilution (50 mL) once the targeted molar mass was reached. The diluted reaction mixture was then acidified with 1.0 M HC1 to pH ⁇ 4, and then purified by dialysis against acidic water (4.0 L, pH ⁇ 5, refreshed 6 times in 3 days). The polymers in their HCl-salt form were collected as white solid after freeze-dry.
  • RNA derived from the Venezuelan Equine Encephalitis Virus VEEV
  • fLuc firefly luciferase
  • eGFP enhanced green fluorescent protein
  • HA hemagglutinin
  • RNA transcripts were synthesized using 1 pg of linearized DNA template in a MEGAScriptTM reaction (Promega, UK) according to the manufacturer’s protocol. Transcripts were then purified by overnight LiCl precipitation at -20 °C, pelleted by centrifugation at 14,000 rpm for 20 min, washed lX with 70% EtOH, centrifuged at 14,000 rpm for 5 min, and then resuspended in UltraPure H 2 0.
  • ScriptCapTM nvG Capping System CellScript, Madison, WI, USA
  • ScriptCaptTM 2’-0-Methyltransferase Kit CellScript, Madison, WI, USA
  • Capped transcripts were then purified by LiCl precipitation as detailed above, resuspended in UltraPure H 2 0 and stored at -80 °C until further use.
  • PEI Polyethylene glycol
  • PABOLs Stock solutions of PEI, PABOLs and saRNA were prepared first by directly dissolving these materials in molecular grade water and stored in fridge.
  • the concentration of the stock solutions are 2.00 pg/ pL (PEI), 0.24 pg/ pL (fLuc Mut Rep RNA) and 5.00 pg/ pL (PABOLs, in vitro studies) or 50 pg/ pL (PABOLs, in vivo studies), respectively.
  • Polyplexes were prepared using two methods: a) ‘direct mixing’ and b) ‘titration’.
  • RNA solution was added to the polymer solution at a rate of 160 pL/min (unless otherwise stated).
  • a series of complex solutions were prepared with the polymer/ saRNA weight ratios ranging from 1/ 1 to 60/ 1.
  • Transfections were performed in HEK293T.17 cells (ATCC, USA) that were maintained in culture in complete Dulbecco’s Modified Eagle’s Medium (cDMEM) (Gibco, Thermo Fisher, UK) containing 10% fetal calf serum (FCS), 5 mg/ mL L-glutamine and 5 mg/ mL penicillin/streptomycin (Thermo Fisher, UK). Cells were plated at a density of 50,000 cells per well in a clear 96 well plate 24 h prior to transfection. For the transfection, the media was completely removed and replace with 50 pL of pre-warmed transfection medium (DMEM with 5 mg/mL L-glutamine).
  • BSO buthionine sulfoximine
  • cells were transfected with varying ratios of PABOL and PEI to saRNA ranging from 10:1 to 450:1 (w/w) according to the above protocol. 24 h after the initial transfection, 20 pL of CellTiter-Blue reagent (Promega, UK) was added to each well and allowed to incubate for 1 h. The plate was then analyzed for absorbance on a FLUOstar Omega plate reader (BMG LABTECH, UK) and normalized to the media control.
  • FLUOstar Omega plate reader BMG LABTECH, UK
  • mice In vivo fLuciferase expression in mice. All animals were handled in accordance with the UK Home Office Animals Scientific Procedures Act 1986 and with an internal ethics board and UK government approved project and personal license. Food and water were supplied ad libitum.
  • IM intramuscularly
  • ID intradermally
  • mice were injected intraperitoneally (IP) with 100 pL of XenoLight RediJect D- Luciferin Substrate (Perkin Elmer, UK) and allowed to rest for 10 min. Mice were then anesthetized using isoflurane and imaged on an In Vivo Imaging System (IVIS) FX Pro (Kodak Co., Rochester, NY, USA) equipped with Molecular Imaging Software Version 5.0 (Carestream Health, USA) for 2 min. Signal from each injection site was quantified using Molecular Imaging software and expressed as relative light units (p/s).
  • IVIS In Vivo Imaging System
  • Surgically resected specimens of human skin tissue were collected at Charing Cross Hospital, Imperial NHS Trust, London, UK. All tissues were collected after receiving signed informed consent from patients, under protocols approved by the Local Research Ethics Committee. The tissue was obtained from patients undergoing elective abdominoplasty or mastectomy surgeries. Tissue was refrigerated until arrival in the laboratory where the subcutaneous layer of fat was removed, and the tissue was excised into 1 cm 2 sections. Explants were incubated at 37 °C with 5% C0 2 in petri dishes with 10 mL of cDMEM. Media was replaced daily.
  • Explants were injected intradermally (ID) using a Micro-Fine Demi 0.3 mL syringe (Becton Dickinson, UK) with 2 pg of eGFP saRNA/pABOL polyplexes in a volume of 100 pL. After three days, skin explants were minced well with scissors and incubated in 3 mL DMEM supplemented with 1 mg/ mL collagenase P (Sigma, UK) and 5 mg/mL dispase II (Sigma, UK) for 4 h at 37 °C on a rotational shaker. Digests were then filtered through a 70 pm cell strainer and centrifuged at 1750 RPM for 5 min.
  • IM intramuscularly
  • ID intradermally
  • mice were culled and the muscle or skin around the injection site was excised and put in 3 mL DMEM supplemented with 1 mg/ mL collagenase P (Sigma, UK) and 5 mg/mL dispase II (Sigma
  • Digests were then filtered through a 70 pm cell strainer and centrifuged at 1750 RPM for 5 min. Cells were then resuspended in 1 mL of FACS buffer (PBS + 2.5.% FCS) at a concentration of 1E7 cells/mL. too pL of cell suspension was added to a FACS tube and stained with Fixable Aqua Live/Dead Cell stain (Thermo Fisher, UK) dilution 1:400 in FACS buffer for 20 min on ice. Cells were then washed with 1 mL of FACS buffer, centrifuged at 1750 rpm for 5 min and resuspended in 250 pL PBS.
  • FACS buffer PBS + 2.5.% FCS
  • Ex vivo fLuciferase expression in human skin explants Human skin tissue was collected and excised as described above. Explants were incubated at 37 °C with 5% C0 2 in petri dishes with 10 mL of cDMEM. Media was replaced daily. Explants were injected intradermally (ID) using a Micro-Fine Demi 0.3 mL syringe (Becton Dickinson, UK) with 2 pg of fLuc saRNA/pABOL polypi exes in a volume of too pL.
  • mice were immunized IM in one hind leg with either 1 or 0.1 pg of HA saRNA formulated with either in vivo jet-PEI®, PABOL-8 (Table 1, #2) or PABOL-100 (Table 1, #8) in a total volume of 50 pL, and boosted after 6 weeks. Blood was collected after 3, 6 and 9 weeks from study onset via tail bleeding, centrifuged at 10,000 rpm for 5 min and then the serum was removed and stored at -8o°C until further use.
  • mice were challenge with XXX pfu of influenza (Cal/09) suspending in 100 uL of PBS. Mice were anesthetized using isoflurane, challenged intranasally (IN), and weighed each day to determine weight loss. According to challenge protocol, mice were culled if they sustained more than three days of 20% weight loss or one day of 75% weight loss.
  • CMTM6 maintains the expression of PD-Li and regulates anti- tumour immunity. Nature 549, 101-105, doi:io.i038/nature23043 htps://www.nai11re.COm/atticles/nature23643#supplementary-information

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Abstract

L'invention concerne des compositions polymères, des nanoparticules et des vaccins comprenant des compositions polymères. L'invention concerne également des utilisations médicales des compositions polymères, des nanoparticules et des vaccins. L'invention concerne en outre des procédés de production des compositions polymères et des nanoparticules.
EP20793432.4A 2019-10-10 2020-10-09 Composition polymère Pending EP4041802A1 (fr)

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Non-Patent Citations (3)

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Title
ELZES M. RACHÈL ET AL: "Disulfide-functional poly(amido amine)s with tunable degradability for gene delivery", JOURNAL OF CONTROLLED RELEASE ELSEVIER, NL, vol. 244, 24 August 2016 (2016-08-24), pages 357 - 365, XP029850306, ISSN: 0168-3659, Retrieved from the Internet <URL:https://www.sciencedirect.com/science/article/pii/S0168365916305545> [retrieved on 20160824], DOI: 10.1016/J.JCONREL.2016.08.021 *
GANG CHEN ET AL: "Fluorination Enhances Serum Stability of Bioreducible Poly(amido amine) Polyplexes and Enables Efficient Intravenous siRNA Delivery", ADVANCED HEALTHCARE MATERIALS, WILEY - V C H VERLAG GMBH & CO. KGAA, DE, vol. 7, no. 5, 27 December 2017 (2017-12-27), pages n/a, XP072461987, ISSN: 2192-2640, Retrieved from the Internet <URL:https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.201700978> [retrieved on 20171227], DOI: 10.1002/ADHM.201700978 *
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